Methods of identifying compounds that modulate igg mediated mast cell activation

ABSTRACT

The present invention relates to methods for identifying compounds capable of modulating Fcg receptor signaling pathway in mast and/or basophil cells. The invention further relates to methods for identifying compounds for use as therapeutic agents in the treatment of disorders related to activation of Fcg receptor signaling cascade.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/402,288, filed Aug. 9, 2002, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to methods of identifying compoundscapable of modulating Fcγ receptor-mediated signaling cascade in mastand basophil cells and identifying compounds for treating disordersrelated to activation via the Fcγ signal transduction pathway. Thepresent invention further relates to methods of identifying compoundscapable of modulating IgE-dependent enhancement of Fcγ receptor-mediatedmast and/or basophil cell activation.

BACKGROUND OF THE INVENTION

Crosslinking of Fc receptors, such as the high affinity receptor for IgE(FcεRI) and/or the high affinity receptor for IgG (FcγRI) activates asignaling cascade in mast, basophil and other immune cells that resultsin the release of chemical mediators responsible for numerous adverseevents. In mast and basophil cells, activation of the FcεRI or FcγRIsignaling cascade leads to the immediate (ie., within 1-3 min. ofreceptor activation) release of preformed mediators of atopic and/orType I hypersensitivity reactions (e.g., histamine, proteases such astryptase, etc.) via the degranulation process. Such atopic or Type Ihypersensitivity reactions include anaphylactic reactions toenvironmental and other allergens (e.g., pollens, insect and/or animalvenoms, foods, drugs, contrast dyes, etc.), anaphylactoid reactions, hayfever, allergic conjunctivitis, allergic rhinitis, allergic asthma,atopic dermatitis, eczema, urticaria, mucosal disorders, tissuedisorders and certain gastrointestinal disorders.

The immediate release of the preformed mediators via degranulation isfollowed by the release and/or synthesis of a variety of other chemicalmediators, including, among other things, platelet activating factor(PAF), prostaglandins and leukotrienes (e.g., LTC4) and the de novosynthesis and release of cytokines such as TNFα, IL-4, IL-5, IL-6,IL-13, etc. These “late stage” mediators are thought to be in partresponsible for the chronic symptoms of atopic and Type Ihypersensitivity reactions, and in addition are chemical mediators ofinflammation and inflammatory diseases (e.g., osteoarthritis,inflammatory bowel disease, ulcerative colitis, Crohn's disease,idiopathic inflammatory bowel disease, irritable bowel syndrome, spasticcolon, etc.), low grade scarring (e.g., scleroderma, increased fibrosis,keloids, post-surgical scars, pulmonary fibrosis, vascular spasms,migraine, reperfusion injury and post myocardial infarction), and siccacomplex or syndrome.

Generally, the major pathway of mast and basophil cell activation, andpathogenesis of allergic diseases, is believed to occur throughIgE-associated signal transduction via FcεRI receptors. Contact with avariety of allergens is correlated with elevated levels of allergenspecific IgE antibodies, and mice with targeted gene disruption of FcεRIα or γ chain display deficits in anaphylactic responses (Galli, S. J.and Lantz, C. S., Allergy, in Fundamental Immunology, 4th Ed., Paul W.E. ed., Lippincott-Raven Publishers, New York (1994)). Moreover,crosslinking of FcεRI by IgE and multivalent antigen or anti-IgEantibodies produces robust degranulation of mast cells and subsequentsynthesis of late stage mediators.

The presence of various Fcγ receptors on mast cells and basophil cells,however, suggests that IgG-dependent activation also participates inallergic responses of these cells and other host defense mechanisms. Forexample, targeted gene disruption of Fcε receptor does not completelyeliminate anaphylactic responses to allergens, and mast cellsdegranulate upon IgG-mediated activation of Fcγ receptor signalingcascade (Okayama, Y. et al. J. Immunol. 164(8):4332-4339 (2000)). Theresponse of mast cells to Fcγ receptor activation, however, is lowerthan those seen for FcεR activation. Resting human mast cells expresslow levels of high affinity FcγR1, and subjecting the cells to receptorcrosslinking conditions does not result in detectable levels ofhistamine release (Okayama et al., supra). In mouse mast cells known toexpress FcγRII and FcγRIII receptors, activation of these Fcγ receptorsresults in release of granule-associated mediators and enzymes, but atlevels below that observed for FcεRI receptor activation (Katz, H. R. etal., J. Immunol. 148(3):868-871 (1992)). The decreased responsiveness ofmast cells to Fcγ receptor activation presents a disadvantage in screensfor compounds that modulate Fcγ receptor-mediated signaling, andconsequently the identification of compounds with therapeuticapplications, particularly those that may distinguish between IgG andIgE-mediated cellular responses. It is known that interferon gamma(IFN-γ) upregulates FcγRI in mast cells, thus providing a method ofenhancing signal transduction by FcγRI (Okayama et al., supra). IFN-γ,however, potentially affects other cellular regulatory mechanisms thatmay lead to changes in the phenotypic character of mast cell populationsused in a screen. The possible pleiotropic effects of exposing mast orbasophil cells to IFN-γ complicate screens for compounds that modulateFcγ signaling pathways. Thus, it is desirable in the art to haveadditional ways of examining Fcγ receptor-mediated signaling in mastand/or basophil cells, in addition to methods using FcγRI upregulationby IFN-γ.

SUMMARY OF THE INVENTION

The present invention relates to methods of identifying compounds thatmodulate Fcγ receptor-mediated signaling pathways. The methods takeadvantage of the ability of IgE antibody to enhance Fcγ dependentactivation of mast cells. These IgE primed mast cells display increaseddegranulation to Fcγ receptor activation, thereby providing mast cellsuseful in identifying compounds that modulate Fcγ mediated signalingpathway. It is to be understood that the methods described herein alsoapply to basophil cells as well.

Accordingly, in one aspect, the present invention provides a method ofidentifying compounds that are capable of modulating Fcγreceptor-mediated signaling in mast and/or basophil cells. The methodgenerally comprises contacting at least one IgE primed mast or basophilcell with a candidate compound in the presence of Fcγ signaling cascadeactivation. The primed cells are made by contacting the cells with IgEantibody, which may be naturally occurring or recombinant IgE;monoclonal or polyclonal; or whole IgE molecule or IgE fragmentscontaining the Fc region. The cell is then evaluated to determinewhether the compound modulates Fcγ receptor-mediated mast and/orbasophil cell activation.

The methods of the present invention may be used to identify compoundswith therapeutic potential for treating or preventing diseases relatedto Fcγ receptor-mediated mast and/or basophil cell activation. Diseasesassociated with mediator release include, by way of example and notlimitation, atopy or anaphylactic hypersensitivity or allergicreactions, allergies, low grade scarring, diseases related to tissuedestruction, often associated with inflammatory reactions.

In a further aspect, the present invention provides for a method ofidentifying a compound capable of modulating IgE-mediated priming ofmast and/or basophil cells. The method generally comprises contacting atleast one mast or basophil cell with a candidate compound and primingthe cell with IgE antibody. The treated cells are activated bystimulating Fcγ receptor signaling cascade to determine whether thecandidate compound modulates IgE priming of the mast or basophil cells.Preferably, the cells are contacted with the candidate compound prior toIgE priming to distinguish effects of the compound on cell priming ascompared to effects on Fcγ receptor-mediated cell activation.

The cells for screening may be isolated from tissues or comprisecultured cells. Preferably for screening purposes, cultured mast orbasophil cells are used because of the ability to generate large numbersof cells, particularly by culturing the mast and basophil cells fromhematopoietic stem cells. Screened mast cells may be of a specificphenotype, such as mucosal mast cells and connective tissue-type mastcells. In humans, musocal mast cells are typicallytryptase-positive/chymase negative while connective tissue-type mastcells are typically tryptase and chymase positive. Any animal can serveas sources for mast and/or basophil cells, including mice and rats andother commonly used experimental animals, to higher animals, such asprimates, particularly human subjects.

The candidate compound can be any compound, including but not limitedto, small organic molecules, saccharides, carbohydrates,polysaccharides, lectins, peptides, proteins, nucleic acids, and thelike. Preferably, the candidate compounds comprise small organicmolecules with a molecular weight of about 100-2500 daltons. The smallorganic molecules may comprise cyclic structures of carbon atoms, orcombination of carbon atoms and heteroatoms, and/or aromatic,polyaromatic, and heteroaromatic structures.

For embodiments of the method using candidate compounds capable oftraversing the cell membrane, the candidate compounds may beadministered to the cell by contacting the cell with the compounds. Inembodiments of the methods in which the candidate compounds can beexpressed or transcribed, such as proteins or RNA, the candidatecompounds are administered to the cell as polynucleotides capable ofexpressing or transcribing the compound in the cell.

The methods of the present invention provide for contacting the mastand/or basophil cells with candidate compounds in the presence of Fcγreceptor signaling activation. Three types of Fcγ receptors are known,including FcγRI, FcγRII, and FcγRIII, and each may be subject toactivation in the present invention. In one aspect, a specific Fcγreceptor signaling pathway is selectively activated. Preferentialactivation of one receptor pathway is done by contacting the mast and/orbasophil cells with antibodies, either whole antibodies or F(ab)₂fragements, directed against the specific receptor, and crosslinking ofthe receptor bound antibody or F(ab)₂ fragment. In one embodiment, theFcγRI receptor is selectively activated.

In the present invention, determining whether a compound is capable ofmodulating Fcγ receptor-mediated mast cell activation is done byevaluating cellular responses downstream of Fcγ receptor activation.These include determining release of preformed mediators by mast orbasophil cell degranulation, or determing synthesis of late phasemediators.

In a final aspect, the present invention provides for kits for carryingout the methods of the invention. In one embodiment, the kit comprisesmast and/or basophil cells, IgE antibodies for priming of the cells,components for activating the cells via the Fcγ receptor signalingpathway, and assay reagents for determining level of cell activation.Additional components may accompany the kits, including, by way ofexample and not limitation, buffers, labels, enzyme substrates, culturemedium, and instructions describing use of the kits.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the analysis of tryptase activity in thesupernatant for stimulated populations of IgE primed mast cells atvarious IgG concentrations (human IgG3) and various dilutions of rabbitanti-human IgG. Five concentrations of IgG were tested at six differentdilutions of anti-IgG antibody in the absence (FIG. 1A) or presence(FIG. 1B) of IL-4 in the priming solution. Controls included activationwith ionomycin or stimulation with media lacking antibody. Addition ofIL-4 to the priming solution did not appear to substantially affectactivation;

FIGS. 2A and 2B show analysis of tryptase activity in the supernatant ofstimulated populations of IgE primed mast cells and at variousconcentrations of IgG (human IgG3) and activated using various dilutionsof rabbit anti-human IgG. Cells were stimulated with either anti-IgGalone (FIG. 2A) or in conjunction with anti-IgE (rabbit anti-human IgE)antibody (FIG. 2B);

FIG. 3 shows plots of FACS-sorted cells immunostained for various Fcγreceptor types without priming (SCF/IL-6) or with priming(SCF/IL-6/IL-4/IgE). Plots left of the vertical line show staining withantibodies directed to three different Fcγ receptor types, includingFcγRI. Plots right of the vertical line show staining with antibodiesagainst human IgE and IgG following incubation with either IgE orvarious types of IgG. IgG4 is known to bind only FcγRI. The data showsthat levels of FcγRI increase in cells primed with IgE;

FIG. 4 shows plots of FACS sorted mast cells immunostained withanti-IgE/G under various priming conditions and incubated with eitherIgE or various types of IgG;

FIGS. 5A-5C show the effects of increasing concentrations of fivedifferent compounds which inhibit degranulation, as measured by tryptaserelease into the media by mast cells following activation with anti-IgE(FIG. 5A), anti-IgG (FIG. 5C), or both anti-IgE/G (FIG. 5B);

FIG. 6A-6E show that each of the inhibitory compounds A-E inhibitsdegranulation similarly, regardless of the mode of activation;

FIG. 7 shows tryptase activity in supernatants of cultured mast cellsactivated under various conditions. Culture conditions are shown on theright side of the figure. Activation conditions are indicated on thelower portion of each bar graph. In the presence of priming with IgE,mast cells show higher levels of tryptase activity upon activation ofFcγ signaling pathway with IgG4 and anti-IgG.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

As used throughout the instant application, the following terms shallhave the following meanings:

“Identifying” in the context of screening assays means determiningwhether a candidate compound unknown to possess a particular property ofinterest possesses the property of interest, as well as confirming thata compound thought or known to possess a particular property of interestpossesses the property of interest.

“Fc Receptor” refers to a member of the family of cell surface moleculesthat binds the Fc portion (containing the specific constant region) ofan immunoglobulin. Each Fc receptor binds immunoglobulins of a specifictype. For example, the Fcα receptor (“FcαR”) binds IgA, the FcεR bindsIgE, and the FcγR binds IgG.

The FcαR family includes the polymeric Ig receptor involved inepithelial tansport of IgA/IgM, the mycloid specific receptor RcαRI(also called CD89), the Fcα/μR and at least two alternative IgAreceptors (for a recent review, see Monteiro and van de Winkel, Annu.Rev. Inmunol, advanced e-publication (2003)). The FcαRI is expressed onneutrophils, eosinophils, moncytes/macrophages, dendritic cells andkupfer cells. The FcαRI inclues one alpha chain and the FcR gammahomodimer that bears an activation motif (ITAM) in the cytoplasmicdomain and phosphorylates Syk kinase.

The FcεR family includes two types, designated FcεRI and FcεRII (alsoknown as CD23). FcεRI is a high affinity receptor (binds IgE with anaffinity of about 10¹⁰M⁻¹) found on mast, basophil and eosinophil cellsthat anchors monomeric IgE to the cell surface. The FcεRI possesses onealpha chain, one beta chain and the gamma chain homodimer discussedabove. The FcεRII is a low affinity receptor expressed on mononuclearphagocytes, B lymphocytes, eosinophils and platelets. The FcεRIIcomprises a single polypeptide chain and does not include the gammachain homodimer.

The FcγR family includes three types, designated FcγRI (also known asCD64), FcγRII (also known as CD32) and FcγRIII (also known as CD16).FcγRI is a high affinity receptor (binds IgG1 with an affinity of10⁸M⁻¹) found on mast, basophil, mononuclear, neutrophil, eosinophil,deudritic and phagocyte cells that anchors monomeric IgG to the cellsurface. The FcγRI includes one alpha chain and the gamma chain dimershared by FcαRI and FcεRI.

The FcγRII is a low affinity receptor expressed on mast cells,neutrophils, monocytes, eosinophils, platelets, and B lymphocytes. TheFcγRII includes one alpha chain, and does not include the gamma chainhomodimer discussed above.

The FcγRIII is a low affinity (binds IgG1 with an affinity of 5×10⁵M⁻¹)expressed on NK, eosinophil, macrophage, neutrophil, and mast cells. Itcomprises one alpha chain and the gamma homodimer shared by FcαRI, FcεRIand FcγRI.

Skilled artisans will recognize that the subunit structure and bindingproperties of these various Fc receptors, cell types expressing them,are not completely characterized. The above discussion merely reflectsthe current state-of-the-art regarding these receptors (see, e.g.,Immunobiology: The Immune System in Health & Disease, 5th Edition,Janeway et al., Eds, 2001, ISBN 0-8153-3642-x, FIG. 9.30 at pp. 371),and is not intended to be limiting with respect to the myriad receptorsignaling cascades.

“Mast cell” refers to specialized, differentiated cells derived fromhematopoietic stem cells. Generally, mast cells are characterized bymetachromatic staining with basic dyes, for example toluidine blue,which bind acidic proteoglycans present in secretory granules. Whenactivated, mast cells degranulate, releasing preformed mediators andenzymes. These substances include biogenic amine histamine; proteaseschymase, tryptase, carboxypeptidase, and cathepsin-G; enzymehexosaminidase; and proteoglycans. In addition, mast cells express anumber of characteristic cell surface molecules, including, by way ofexample and not limitation, FcεRI and FcγRI.

Mast cells are heterogeneous group of cells, and characterized in theart by differing tissue distribution, morphology, histochemistry,mediator content, and response to activation stimuli (Welle, M., J.Leukoc. Biol. 61:233-245 (1997)). Typically, one major basis ofdifferentiating mast cells is the presence of granule-associatedproteases, chymotrypsin-like and trypsin-like proteases (i.e., chymaseand tryptase). In humans, mast cells of the “mucosal,” “mucosal type,”“airway,” or “airway type” refers to mast cells that are typical of mastcells present in lung or intestinal mucosa. Mucosal mast cells arecharacterized by specific binding of high affinity IgE and granulerelease upon cross-linking of IgE receptors with anti-IgE antibody; byhaving granules containing histamine; by absence of chymase; and bypresence of tryptase. Generally, the mucosal mast cells exhibit atryptase positive/chymase negative phenotype. In addition, the mucosalmast cells are typically positive for surface expression of highaffinity IgE receptors, CD54 and CD117, and negative for or haveinsignificant surface expression of CD15, CD34, CD25 and CD11b.

Mast cells of the “connective tissue-type” or “cutaneous” or “serosal”refers to mast cells that are typical of mast cells present inconnective tissues, for example, skin, lymph nodes and intestinalsubmucosa. Connective tissue-type mast cells are characterized by highaffinity binding of IgE and granule release upon cross-linking of IgEreceptors with anti-IgE antibody, by having granules containingrelatively high levels of histamine as compared to mucosal mast cells;and by presence of both chymase and tryptase. Generally, the connectivetissue-type mast cells of the invention exhibit a tryptase/chymasepositive phenotype. In addition, connective tissue-type mast cells aretypically positive for surface expression of high affinity IgE receptor,CD54, and CD117, and negative for or have insignificant surfaceexpression of CD15, CD34, CD25 and CD11b.

The classifications of mast cells in the foregoing are not to be appliedrigidly and are not intended to limit the present invention. Thoseskilled in the art will understand the limitations of suchcategorizations and comprehend the scope of mast cells as used herein.

“Basophil cell” refers to specialized cells that originate fromhematopoietic stem cells and are heavily granulated. Basophilssynthesize many of the same mediators as mast cells, and express thesame high affinity Fcε receptors. Thus, basophils also mediate immediatehypersensitivity reactions to antigen. Additionally, basophilsparticipate in cell-mediated hypersensitivity. Unlike mast cells,basophils mature in the bone marrow and circulate in the blood, fromwhich they are recruited to tissue sites of inflammation.

Basophils are characterized by specific binding of high affinity IgE andgranule release upon cross-linking of IgE receptors with anti-IgEantibody, and by having granules containing, for example, histamine andheparin. Basophils are typically positive for surface expression ofCD11b, CD13 and CD25, but negative for expression of CD14 and CD117 (seeValent, P. et al., Adv. Immunol. 52:335-339 (1992); Agis, H. et al.,Immunology 87:535-43 (1996), hereby incorporated by reference). Thus, inone embodiment, the basophils of the invention are CD11b-positive,CD13-positive, and CD25-positive. In another embodiment, the basophilsof the invention are CD14-negative and CD117-negative. The basophils mayalso be CD54 positive.

“IgE” refers to an antibody with an Fc region corresponding to the heavychain IgE isotype. IgE may be a naturally occuring antibody, an antibodygenerated by recombinant methods, or a chimeric antibody with an IgEheavy chain of one animal and a light chain of another animal.Preferably, the IgE has the Fc region corresponding to the IgE presentin the animal from which the mast cells are isolated. Thus, for humanmast cells, the IgE is preferably a human IgE. The Fc region of IgEantibodies binds to high affinity FcεRI receptors present on mast cellsand basophil cells.

“IgG” refers to an antibody with an Fc region corresponding to the heavychain of the IgG isotype. IgGs are generally classified into foursubclasses IgG1, IgG2, IgG3 and IgG4. These subclasses aredistinguishable based on numbers of disulfide linkages, molecularweight, abundance in serum, ability to activate complement, antibodydependent cell-mediated cytotoxicity, and binding to various FcγRreceptors (Galli, S. J. and Lantz, C. S., Allergy in FundamentalImmunology, 4th Ed., Paul W. E. ed., Lippincott-Raven Publishers, NewYork (1994)). For example, IgG1 and IgG4 bind to high affinity FcγRI. Aswith other immunoglobulin isotypes, IgGs may be a naturally occuringantibody, an antibody generated by recombinant methods, or a chimericantibody with an IgG heavy chain of one animal and a light chain ofanother animal.

“Primed mast cell” refers to a mast cell displaying enhanced activationcompared to an unprimed mast cell. Enhanced or increased activationrefers to elevated cellular responses to activating stimuli, such asIgG-mediated activation of Fcγ receptors. In the present invention, theprimed mast cells may show degranulation at lower concentrations of IgG,show increases in the amount and duration of release of granulecontents, and display changes in other downstream cellular eventsassociated with activation of Fcγ receptor signaling pathway. “Primedbasophil cell” as used herein is defined similarly as primed mast cells.

A compound that “modulates Fcγ receptor-mediated signaling” or that“modulates Fcγ signaling cascade” has the ability to change or alterdownstream cellular events resulting from activation of the signalingpathway. The change may be monitored at the RNA level, for example byquantifying induced downstream transcription products, or at the proteinlevel, for example by quantifying the amount or activity of induceddownstream translation products. Alternatively, the downstream cellularevent is a change in cellular physiology, for example degranulation orrelease of lipid mediators and cytokines.

“Fc Receptor-Mediated Degranulation” or “Fc Receptor-InducedDegranulation” refers to degranulation that proceeds via an Fc receptorsignal transduction cascade initiated by crosslinking of an Fc receptor.

“IgE-Induced Degranulation” or “FcεRI-Mediated Degranulation” refers todegranulation that proceeds via the IgE receptor signal transductioncascade initiated by crosslinking of FcεRI-bound IgE. The crosslinkingmay be induced by an IgE-specific allergen or other multivalent bindingagent, such as an anti-IgE antibody. In mast and/or basophil cells, theFcεRI signaling cascade leading to degranulation may be broken into twostages: upstream and downstream. The upstream stage includes all of theprocesses that occur prior to calcium ion mobilization. The downstreamstage includes calcium ion mobilization and all processes downstreamthereof Compounds that inhibit FcεRI-mediated degranulation may act atany point along the FcεRI-mediated signal transduction cascade.Compounds that selectively inhibit upstream FcεRI-mediated degranulationact to inhibit that portion of the FcεRI signaling cascade upstream ofthe point at which calcium ion mobilization is induced. In cell-basedassays, compounds that selectively inhibit upstream FcεRI-mediateddegranulation inhibit degranulation of cells such as mast or basophilcells that are activated or stimulated with an IgE-specific allergen orbinding agent (such as an anti-IgE antibody) but do not appreciablyinhibit degranulation of cells that are activated or stimulated withdegranulating agents that bypass the FcεRI signaling pathway, such as,for example the calcium ionophores ionomycin and A23187.

“IgG-Induced Degranulation” or “FcγR-mediated Degranulation” refers todegranulation that proceeds via the Fcγ signal transduction cascade,typically initiated by crosslinking of FcγR-bound IgG. The crosslinkingmay be induced by an IgG-specific allergen or another multivalentbinding agent, such as an anti-IgG or fragment antibody. Like the FcεRIsignaling cascade, in mast and basophil cells the Fcγ signaling cascadealso leads to degranulation which may be broken into the same twostages: upstream and downstream. Similar to FcεRI-mediateddegranulation, compounds that selectively inhibit upstream Fcγ-mediateddegranulation act upstream of the point at which calcium ionmobilization is induced. In cell-based assays, compounds thatselectively inhibit upstream Fcγ receptor-mediated degranulation inhibitdegranulation of cells such as mast or basophil cells that are activatedor stimulated with an IgG-specific allergen or binding agent (such as ananti-IgG antibody or fragment) but do not appreciably inhibitdegranulation of cells that are activated or stimulated withdegranulating agents that bypass the Fcγ signaling pathway, such as, forexample the calcium ionophores ionomycin and A23187.

“Ionophore-Induced Degranulation” or “Ionophore-Mediated Degranulation”refers to degranulation of a cell, such as a mast or basophil cell, thatoccurs upon exposure to a calcium ionophore such as, for example,ionomycin or A23187.

“Allergen” refers to antigens capable of binding to IgE or IgGantibodies and mediating activation of mast and/or basophil cells.Allergens comprise various compounds that bind to cognate antibodies andactivate mast and/or basophil cells. These include, by way of exampleand not limitation, pollen grains, environmental particulates (e.g. dustmites), proteins associated with foreign particles or foods, insectvenoms, chemicals (e.g., food preservatives, disinfectants,insecticides, etc.), pharmaceutical agents (e.g., antibiotics, steroids,non-steroidal anti-inflammatory drugs, anesthetics, etc.), and the like.

Fc Receptor Signaling Cascades and Priming of Cells with IgE

The present invention relates to methods of identifying compoundscapable of modulating Fcγ receptor signaling cascades involved indegranulation of mast and/or basophil cells. Both mast and basophilcells play a central role in allergen-induced disorders, including, forexample, allergic rhinitis and asthma. Upon exposure allergens, whichmay be, among other things, pollen or parasites, allergen-specificantibodies are synthesized by B-cells activated by IL-4 (or IL-13).These allergen-specific IgE antibodies bind to the high affinity FcεRIwhile IgG antibodies bind Fcγ receptors. Upon binding of antigen, thereceptors are cross-linked and the IgE or IgG receptor signaltransduction pathway is activated, which leads to degranulation of thecells and consequent release and/or synthesis of a host of chemicalmediators, including histamine, proteases (e.g., tryptase and chymase),lipid mediators such as leukotrienes (e.g., LTC4), platelet-activatingfactor (PAF) and prostaglandins (e.g., PGD2), and a series of cytokines,including TNF-α, IL-4, IL-13, IL-5, IL-6, IL-8, GMCSF, VEGF and TGF-β.The release and/or synthesis of these mediators from mast and/orbasophil cells accounts for the early and late stage responses inducedby allergens, and is directly linked to downstream events that lead to asustained inflammatory state.

The molecular events in the FcεRI and FcγR signal transduction pathwaysthat lead to release of preformed mediators via degranulation andrelease and/or synthesis of other chemical mediators are well-known.Generally, the FcεRI is a heterotetrameric receptor composed of anIgE-binding alpha-subunit, a beta subunit, and two gamma subunits (gammahomodimer). Cross-linking of FcεRI-bound IgE by multivalent bindingagents (including, for example IgE-specific allergens or anti-IgEantibodies or fragments) induces the rapid association and activation ofthe Src-related kinase Lyn. Lyn phosphorylates immunoreceptortyrosine-based activation motifs (ITAMS) on the intracellular beta andgamma subunits, which leads to the recruitment of additional Lyn to thebeta subunit and Syk kinase to the gamma homodimer. Thesereceptor-associated kinases, which are activated by intra- andintermolecular phosphorylation, phosphorylate other components of thepathway, such as the Btk kinase, LAT, and phospholipase C-gammaPLC-gamma. Activated PLC-gamma initiates pathways that lead to proteinkinase C activation and Ca²⁺ mobilization, both of which are requiredfor degranulation. FcεRl cross-linking also activates the three majorclasses of mitogen activated protein (MAP) kinases, i.e. ERK1/2, JNK1/2,and p38. Activation of these pathways is important in thetranscriptional regulation of proinflammatory mediators, such as TNF-αand IL-6, as well as the lipid mediator leukotriene CA (LTC4).

The Fcγ receptor signaling cascade, particularly FcγRI, is believed toshare some common elements with the FcεRI signaling cascade.Importantly, like FcεRI, the FcγRI includes a gamma homodimer that isphosphorylated and recruits Syk, and like FcεRI, activation of the FcγRIsignaling cascade leads to, among other things, degranulation. Other Fcreceptors that share the gamma homodimer include, but are not limitedto, FcαRI and FcγRIII.

In the present invention, the observation that contacting mast cellswith IgE enhances activation mediated by the Fcγ signal transductionpathway, particularly FcγRI, provides a way of generating mast cellsuseful in screens for identifying compounds that modulate the Fcγreceptor signal transduction pathway, and consequently compounds fortreating various disorders related to mast cell activation. In contrastto activation of mast cells by IgE antibody, priming of mast cells withIgE does not appear to require crosslinking of IgE bound to the FcεRIreceptor. Thus, IgE-mediated degranulation and mediator release is not aprerequisite for priming mast cells with the IgE antibody. It is to beunderstood that other cells containing IgE and IgG receptors, such asbasophil cells, may be also subjected to the priming step to enhanceactivation of the Fcγ receptor-mediated signaling pathway.

Accordingly, the present invention provides for methods of priming amast and/or basophil cell by contacting the cell with IgE antibody andusing the cells in methods of identifying compounds that modulate Fcγreceptor-mediated signal transduction pathways. In one aspect, themethods for identifying compounds that modulate Fcγ receptor signalingpathway comprise contacting at least one IgE primed mast and/or basophilcell with a candidate compound in the presence of Fcγ receptor signalingcascade activation, and determining whether the candidate compoundmodulates the Fcγ receptor-mediated signaling pathway.

In one aspect, by comparing cell activation in the presence and absenceof the candidate compound, modulation of Fcγ signaling cascade can bedetermined. In one embodiment, the modulation is inhibition of Fcγreceptor-mediated cell activation. This effect may be seen as a decreasein cellular responses to Fcγ activation. As provided in detail below,the response of the mast or basophil cell to Fcγ receptor activation isdetermined by detecting cellular responses downstream of the Fcγreceptor signaling cascade. Downstream cellular events include releaseof preformed mediators, for example histamine and serotonin;proteoglycans; and proteases such as serine proteases andcarboxypeptidase. In a further embodiment, the cellular downstreamevents are late phase events, which can be evaluated by measuring, byway of example and not limitation, synthesis and release of lipidmediators, and synthesis and release of cytokines.

In a further embodiment, the present invention provides for methods ofidentifying compounds capable of modulating priming of mast and/orbasophil cells by IgE. Generally, the method comprises contacting atleast one mast or basophil cell with a candidate compound and IgEantibody. The Fcγ signaling cascade is activated, and the modulation ofpriming determined by evaluating levels of cell activation.

Increases or decreases of IgE-mediated cell priming is measured.Attenuated or a decreased level of Fcγ receptor-mediated cell activationwill indicate an inhibitory effect of the compound on priming. Increasedlevel of Fcγ receptor-mediated cell activation will indicate astimulatory effect of the compound on priming. By comparing Fcγreceptor-mediated mast or basophil cell activation in presence andabsence of candidate compound, modulators of IgE priming can beidentified. Contacting or administering the compound before Ig primingwill assist in distinguishing between effects of the compound on primingversus cell activation. Furthermore, effect on priming and activation isalso distinguishable by examining changes in surface markers,particularly the level of IgG receptors expressed on the cell surface,which in the present invention is found to increase with IgE priming.

Preferably, populations of mast cells are used to screen large numbersof candidate compounds in the methods described herein, particularlywhen high throughput screens are carried out. The population of mastand/or basophil cells may contain 10⁶-10¹¹ cells. Thus, the populationsof cells used for screening may comprise about 10⁶ cells, preferably 10⁹cells, more preferably 10¹⁰ cells, and most preferably 10¹¹ cells.

Sources of Mast and Basophil Cells for Screening

The mast cells and basophil cells for use in the present invention maybe obtained in a variety of ways known to the skilled artisan. Theseinclude isolation of cells from tissues, or culturing the cells fromprogenitor cells by suitable treatment with growth factors andcytokines. Isolation of mast and basophil cells from tissues typicallyinvolves enzyme-mediated tissue dispersion and subsequent enrichment bygradient sedimentation, such as single Percoll gradient or multiplePercoll gradients (Patella, V. et al., J Immunol. 154(6):2855-2865(1995); Stankiewicz, M., et al, Int J Parasitol. 24(2):307-309 (1994)).In another aspect, cell sorting is used to enrich and/or purify thecells. Cellular parameters useful for obtaining enriched population ofmast or basophil cells include density, granularity, size, andautofluorescence. Other methods include positive/negative selection byfluorescence activated cell sorting (FACS) using antibodies directed tomast or basophil cell markers, preferably cell surface molecules.Alternatively, negative selection by selective removal of non-mast ornon-basophil cells by killing with complement or selective attachment tosubstrates offer alternative basis for preparing enriched or purifiedpopulation of cells (Valent, P. and Bettelheim, P., Adv. Immunol.52:333-423 (1992)).

In another aspect, mast cells and/or basophil cells are generated invitro from hematopoietic stems cells using media containing combinationsof growth factors and cytokines. Typically, umbilical cord blood, bonemarrow, or fetal liver serves as sources of hematopoietic stem cells.Various in vitro methods for expanding mast or basophil cells are knownto the skilled artisan. For example, mast cells may be generated in asingle step by contacting CD34+ positive stem cells with variouscombinations of stem cell factor (SCF), flt-3 ligand, and cytokines thatexpand populations of progenitor cells and also induce differentiationinto various mast cell types. For example, SCF and IL-6 induces stemcells to expand and differentiate into a population of mast cellsdisplaying predominantly tryptase positive cells and a minor percentageof chymase positive cells (Saito, H. et al., Int. Arch. Allergy Immunol.107:63-65 (1995)). Additional extracellular factors used to expandcultured mast cells include prostaglandin and thrombopoietin (Saito, H.et al., J Immunol. 157(1):343-350 (1996); Gilmore, G. L. et al., ExpHematol. 28(11):1297-1305 (2000)).

Preferably, the cultured mast or basophil cells are expanded from apopulation of progenitor cells, as described in copending U.S.application Ser. No. 10/053,355, filed Nov. 8, 2001, the entire contentsof which is incorporated herein by reference. Generally, a population ofCD34+ positive cells is contacted with SCF and flt-3 ligand to generatea population of progenitor cells capable of differentiating into mast orbasophil cells. Subsequently, the expanded population of progenitorcells is contacted with SCF and a cytokine suitable for inducingdifferentiation of the expanded progenitor cells into mast or basophilcells. Presence of cytokine IL-6 induces differentiation into mucosalmast cells; presence of IL-4 induces differentiation into connectivetissue-type mast cells; and presence of IL-3 induces differentiationinto basophil cells.

In another aspect, the type of mast cells used in the assay will have adefined phenotype. Thus, in one embodiment, the mast cells in thescreens may comprise mucosal mast cells, which for human mast cellstypically have a tryptase positive and chymase negative phenotype. Inanother embodiment, the mast cells in the screens may compriseconnective tissue-type mast cells, which for human mast cells typicallyhave tryptase and chymase positive phenotype. The known heterogeneity ofmast cells with regard to granule content and tissue distributionsuggests that mast cells differ with respect to their role in differentallergic and inflammatory responses. Thus, mucosal type mast cellstypical of the lung and intestinal submucosa, and connective tissue-typemast cells typical of the skin, intestinal submucosa, and lymph nodes,may provide mast cell types that respond differently to candidatecompounds. This allows identification of compounds selective forparticular types of allergic and inflammatory responses.

The origin of the mast or basophil cells used in the assay will depend,in part, on the desired use for the compounds and will be apparent tothose of skill in the art. For example, if the compounds will be used totreat or prevent a particular disease in humans, a convenient source ofmast or basophil cells is a human or other animal that constitutes anaccepted or known clinical model for the particular disease. Thus,depending upon the particular application, the mast or basophil cellsmay be derived from a wide variety of animal sources, ranging from, forexample, lower mammals such as mice and rats, to dogs, sheep and othermammals commonly employed in clinical testing, to higher mammals such asmonkeys, chimpanzees and apes, to humans. Specific examples of cellssuitable for carrying out the in vitro assays include, but are notlimited to, rodent or human mast cells, primary mouse mast cells (suchas bone marrow-derived mouse mast cells “BMMC”) and primary human mastcells isolated from cord blood (“CHMC”) or other tissues such as lung(see, e.g., Demo, S. D. et al., Cytometry 36(4):340-348 (1999)).

In accordance with the above, the mast or basophil cells may be isolatedfrom animals having variations or defects in processes related to mastor basophil cell activation. These may be naturally occurring geneticdefects or allelic variants in an animal population, or animalsgenetically engineered to have targeted disruptions or misexpression ofgenes involved in mast or basophil cell activation (see, e.g., Matsuda,H et al., Int Immunol. 9(3):461-466 (1997); Li, A. et al., Clin Sci(Lond). 93(3):279-286 (1997); and Dombrowicz, D. et al., Immunity.8(4):517-529 (1998)). For example, animals carrying disruptions ormutations in Kit receptor may be used (Galli, S. J. et al., Ann. N.Y.Acad. Sci. 664:69-98 (1992)).

Priming and Activation of Mast or Basophil Cells

Mast or basophil cells, isolated from tissues or produced in culture,are contacted with IgE to prime the cells, which renders the cellsusceptible to Fcγ receptor-mediated activation. A variety of IgEantibodies are useful for this purpose. The IgE antibodies may bepolyclonal or monoclonal, or comprise the Fc portion of IgE preparedrecombinantly or by proteolysis of IgE molecules. The IgE may bepreparations of naturally occurring IgE; IgE made recombinantly;chimeric antibodies containing Fc region of one animal species and lightchain of another animal species; or an antibody of one species made inanother animal species, such as completely “humanized” antibodies madein mouse. Generally, chimeric antibodies are produced by cellsengineered with variable heavy chain (VH) and variable light chain (VL)gene segments of the desired specificity from one animal species andspliced to constant heavy chain (CH) and constant light chain (CL) genesegments of another animal species, such as human (Morrison, S. L. etal., Proc. Natl Acad. Sci. USA 81:6851-6866 (1984); Neuberger, M. S. etal., Nature 312:604-608 (1984); Takeda, S. et al., Nature 314:452-454(1985); Boss, U.S. Pat. No. 4,816,397; Cabilly, U.S. Pat. No.4,816,567). In some cases, whole antibodies, monoclonal or polyclonal,of one animal may be made by tranferring the complement of its antibodygenes (e.g., VH, VL, CH and CL) into cells of another animal species inwhich the endogenous antibody genes have been inactivated or deleted.Various methods are known in the art for generating chimeric antibodies,including, but not limited to, phage display libraries (Vaughan, T. J.,Nature Biotechnol. 16(6):535-539 (1998)), and cloning and expressingantibody gene segments in mammalian or plant cells (Shearman, C. W. etal., J. Immunol. 146(3):928-935 (1991); Norderhaug, L. et al., J.Immunol Methods. 204(1):77-87 (1997); Luiten, R. M. et al., HumAntibodies 8(4):169-180. (1997)). Transgenic animals whose antibodygenes have been replaced by antibody genes of another animal species aredescribed in Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA97(2):722-727 (2000) and Lonberg, N. et al., Nature 368:856-859 (1994)).

Generally, although IgE antibodies of one species may be used to primemast or basophil cells of another animal species, preferably the IgEantibodies used for priming have the Fc region derived from the sameanimal species from which the cells are obtained. For example, humanmast or basophil cells are primed with human IgE antibodies or withchimeric antibodies containing a human Fc region, while mouse mast orbasophil cells are primed with mouse IgE antibodies or with chimericantibodies containing a mouse Fc region. The cells are contacted withIgE at a sufficient concentration and incubation time to enhance Fcγreceptor-mediated cell activation. Generally, an IgE concentration of upto about 5 ug/ml may be used, with incubation periods ranging from about3 to about 7 days. By way of example and not limitation, priming ofcultured human mast cells is accomplished in culture with about 200ng/ml IgE for about 3 days. Mast cells treated under such conditionsremain primed for about 3 to about 8 days. By contacting mast orbasophil cells with different IgE concentrations and incubation timesand subsequent evaluation of Fcγ receptor-mediated cell activation, theskilled artisan can determine the range of effective conditions requiredfor priming.

Once primed, the cells are activated by various compounds capable ofactivating Fcγ receptor-mediated signaling pathway. Preferably, to limitcomplications from activation of FcεRI receptor-mediated signaling, Fcγreceptors may be selectively activated. In one aspect, activation isthrough use of agents that crosslink Fcγ receptors. Typically, receptorcrosslinking and corresponding activation of the signaling cascade isachieved by combination of an IgG antibody and a cognate anti-IgGantibody capable of crosslinking the receptor bound IgG molecule. Theseanti-IgG antibodies include, by way of example and not limitation, ananti-IgG antibody directed against the heavy and light chain of the IgGantibody. Alternatively, the IgG may comprise a chimeric proteincontaining an Fc region linked to a heterologous protein in which thecrosslinking antibody reacts with the heterologous portion.

Another method of activating mast cells comprises contacting the cellswith an antibody raised directly against the extracellular region of Fcγreceptor and treating the cells with antibodies directed against theanti-Fcγ receptor antibody. The anti-anti Fcγ antibody causes additionalcrosslinking interactions which facilitate activation of the signalingpathway. In a variation of this technique, contacting the cells withF(ab)₂ fragments obtained from anti-Fcγ receptor antibodies and reactingwith an antibody directed against the F(ab)₂ fragment may also produceeffective receptor crosslinking and subsequent activation of signaltransduction. Though use of anti-receptor antibodies or F(ab)₂ fragmentsraised against a specific type of Fcγ receptor (e.g., FcγRI, FcγRII, orFcγRII), specific Fcγ signaling pathways may be activated and examined.

In another aspect, activation of Fcγ receptor signaling pathway isachieved with IgG antibodies directed against a hapten, for exampledinitrophenol (DNP). Mast cells contacted with anti-hapten IgG aretreated with a protein carrier conjugated to a plurality of thespecified hapten (e.g., DNP-bovine serum albumin), thus resulting inreceptor crosslinking and subsequent activation of the Fcγ receptorsignaling cascade. In place of protein carriers, other substrates withattached haptens may be used, including, by way of example and notlimitation, dextran or latex beads. In a further aspect, activation isinduced by use of an IgG antibody reactive with a specific allergen.Various allergens known to react with IgG antibodies include, by way ofexample and limitation, lactalbumin and lactaglobulin in cow's milk(Jarvinen, K. M., Int. Arch. Allergy Immunol. 126(2):111-118 (2001));birch pollen (Denepoux, S. et al., FEBS Lett. 465(1):39-46 (2000); grasspollen (Michils, A. et al., Clin Exp Allergy. 29(6):832-839 (1999); andegg white ovomucoid (Zhang, J. W. et al., Biochem. Biophys. Res. Commun.253(1):124-127 (1998). As the foregoing illustrates, suitable Fcγreceptor activating agents will be apparent to those of skill in theart.

Assays for Determining Mast or Basophil Cell Activation and Fcγ ReceptorSignaling

The primed mast or basophil cells may be subjected to any screeningtechnique known in the art for determining whether compounds modulateFcγ receptor signaling. Suitable assays for modulation of Fcγreceptor-mediated degranulation are similar to those used for detectingdegranulation via the Fcε receptor pathway. In one typical assay, theamount of a chemical mediator or other chemical agent released and/orsynthesized as a consequence of activating the Fcγ signaling cascade maybe quantified using standard techniques and compared to the amount ofthe mediator or agent released from control cells (i.e., cells that arestimulated but that are not exposed to test compound).

As will be recognized by skilled artisans, the mediator or agentquantified is not critical. The only requirement is that it be amediator or agent released and/or synthesized as a consequence ofinitiating or activating the Fc receptor signaling cascade. For example,activation of the Fcγ signaling cascade in mast and/or basophil cellsleads to numerous downstream events. For example, activation of the Fcγsignal cascade leads to the immediate release (i.e., within 1-3 min.following receptor activation) of a variety of preformed chemicalmediators and agents via degranulation. Thus, in one embodiment, themediator or agent quantified may be specific to granules (i.e., presentin granules but not in the cell cytoplasm generally). Examples ofgranule-specific mediators or agents that can be quantified to determineand/or confirm the activity of a candidate compound include, but are notlimited to, granule-specific enzymes such as hexosaminidase, tryptase,and chymase, and granule-specific components such as histamine andserotonin. Assays for quantifying such factors are well-known, and inmany instances are commercially available. For example, tryptase and/orhexosaminidase release may be quantified by incubating the cells withcleavable substrates that fluoresce upon cleavage and quantifying theamount of fluorescence produced using conventional techniques. Suchcleavable fluorogenic substrates are commercially available. Forexample, the fluorogenic substrates Z-Gly-Pro-Arg-AMC(Z=benzyloxycarbonyl; AMC=7-amino-4-methylcoumarin; BIOMOL ResearchLaboratories, Inc., Plymouth Meeting, Pa. 19462, Catalog No. P-142) andZ-Ala-Lys-Arg-AMC (Enzyme Systems Products, a division of ICNBiomedicals, Inc., Livermore, Calif. 94550, Catalog No. AMC-246) can beused to quantify the amount of tryptase released. The fluorogenicsubstrate 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (Sigma, St.Louis, Mo., Catalog No. 69585) can be used to quantify the amount ofhexosaminidase released. Histamine release may be quantified using acommercially available enzyme-linked immunosorbent assay (ELISA) such asImmunotech histamine ELISA assay #IM2015 (Beckman-Coulter, Inc.). Any ofthese assays may be used to determine or confirm the activity ofcandidate compounds.

Degranulation is only one of several responses initiated by the Fcγsignaling cascade. In addition, activation of the signaling pathwayleads to the de novo synthesis and release of cytokines and chemokinessuch as IL-4, IL-5, IL-6, TNF-α, IL-13 and MIP1-α, and release of lipidmediators such as leukotrienes (e.g., LTC4), platelet activating factor(PAF) and prostaglandins. Accordingly, the candidate compounds may alsobe assessed for activity by quantifying the amount of one or more ofthese mediators released and/or synthesized by activated cells.

Unlike the granule-specific components discussed above, these “latestage” mediators are not released immediately following activation ofthe Fcγ signaling cascade. Accordingly, when quantifying these latestage mediators, care should be taken to insure that the activated cellculture is incubated for a time sufficient to result in the synthesis(if necessary) and release of the mediator being quantified. Generally,PAF and lipid mediators such as leukotriene C4 are released 3-30 min.following Fcγ activation. The cytokines and other late stage mediatorsare released approximately 4-8 hrs after receptor activation. Incubationtimes suitable for a specific mediator will be apparent to those ofskill in the art. Specific guidance and assays are provided in theExamples section.

The amount of a particular late stage mediator released may bequantified using any standard technique. In one embodiment, theamount(s) may be quantified using ELISA assays. ELISA assay kitssuitable for quantifying the amount of TNFα, IL4, IL-5, IL-6 and/orIL-13 released are available from, for example, Biosource International,Inc., Camarillo, Calif. 93012 (see, e.g., Catalog Nos. KHC3011, KHC0042,KHC0052, KHC0061 and KHC0132). ELISA assay kits suitable for quantifyingthe amount of leukotriene C4 (LTC4) released from cells are availablefrom Cayman Chemical Co., Ann Arbor, Mich. 48108 (see, e.g., Catalog No.520211).

In addition to the FcεRI or FcγR degranulation pathways discussed above,degranulation of mast and/or basophil cells can be induced by otheragents. For example, ionomycin, a calcium ionophore that bypasses theearly FcεRI or FcγR signal transduction machinery of the cell, directlyinduces a calcium flux that triggers degranulation. Activated PLCγinitiates pathways that lead to, among other things, calcium ionmobilization and subsequent degranulation. This Ca²⁺ mobilization istriggered late in the Fc receptor signal transduction pathway. Asmentioned above, ionomycin directly induces Ca²⁺ mobilization and a Ca²⁺flux that leads to degranulation. Other ionophores that inducedegranulation in this manner include A23187. The ability ofgranulation-inducing ionophores such as ionomycin to bypass the earlystages of the Fcγ receptor signaling cascades may be used as a counterscreen to identify active compounds of the invention that specificallyexert their degranulation-inhibitory activity by blocking or inhibitingthe early Fcγ receptor signaling cascades, as discussed above. Compoundswhich specifically inhibit such early Fcγ receptor-mediateddegranulation inhibit not only degranulation and subsequent rapidrelease of histamine, tryptase and other granule contents, but alsoinhibit the pro-inflammatory activation pathways causing the release ofTNFα, IL-4, IL-13 and the lipid mediators such as LTC4. Thus, compoundswhich specifically inhibit such early Fcγ receptor-mediateddegranulation block or inhibit not only acute atopic or Type Ihypersensitivity reactions, but also late responses involving multipleinflammatory mediators.

Other suitable methods for determining Fcγ receptor-mediated mast cellactivation include examining the biochemical signaling reactions thatoccur upon Fcγ signal transduction. This includes determining thephosphorylation states of kinases and kinase substrates involved in Fcγreceptor signal transduction. In one aspect, the phorphorylation of theITAMs present on Fcγ receptor gamma chains may be examined. These motifsare phosphorylated by tyrosine kinase Lyn and may be detected byantibodies directed to phosphorylated ITAM sequences, orimmuneprecipitation of Fcγ receptor in cells exposed to radioactivesubstrate ATP. It is also known that tyrosine kinases Lyn and Syk areautocatalytic and are phosphorylated upon activation. Thus,phosphorylation states of these kinases may be similarly determined.

In yet another embodiment, the assays for mast cell activation may uselipophilic styryl dyes such as RH414, RH795, FM1-43, and FM 4-64 (seeU.S. application Ser. No. 10/053,355, incorporated herein by reference).These lipophilic dyes reversibly bind the outer leaflet of cellmembranes and become trapped in endocytic vesicles following vesiclerecycling. Upon degranulation, the dyes are released into the mediumresulting in loss of dye from the cells. Because the dyes fluoresce moreintensely in the lipid environment of the endocytic vesicle, thepresence of dye in the cells provides a measure of degranulation (Betz,H. et al., Curr. Opin. Neurobiol. 6:365-371 (1996); Haugland, P. T.,Handbook of Fluorescent Probes and Research Products, 6th Ed, (1996)(see Chapter 17), incorporated herein by reference.

Another dye-based assay useful for measuring degranulation followingcell activation are flurophores linked to weak base. These dyes are cellmembrane permeant, and it is theorized that the dyes are protonated inthe acidic environment of exocytic granules, which results in retentionof the dye in the vesicle. Cell activation results in release of the dyefrom the cell. Thus, changes in dye release will reflect changes in cellactivation. Suitable dyes include LYSOTRACKER® red, LYSOTRACKER® green,and LYSOTRACKER® blue. (Haugland, P. T., supra, Chapter 17; Hafler T. etal., Cell Calcium 19(2):157-165 (1996)).

In yet a further embodiment, the assay for determining mast or basophilcell activation uses the ability of Annexin V to bind exocytic granulesexposed to the external environment (Demo, S. D., Cytometry.36(4):340-348 (1999), incorporated herein by reference). Followingdegranulation, mast cells show an increase in Annexin bindingproportional to the time and intensity of degranulation. Consequently,changes in Annexin binding provide a measure of mast or basophil cellactivation. Annexin is commercially available and may be directlylabeled with a fluorophore, such as fluorescein isothiocyanate (FITC),TRITC, CY5, or other fluorescent compounds known in the art.Alternatively, the Annexin contains a first label, such as a ligand(e.g., biotin), and a cognate binding partner (e.g., streptavidin)labeled with a detectable molecule (e.g., fluorophore, enzyme, etc.) isused to detect presence of the first label. It is understood that theseand other assays for measuring cell activation are well known to theskilled artisan.

Candidate Compounds and Screening of Compounds

The compounds screened can range from small organic molecules to largepolymers and biopolymers, and can include, by way of example and notlimitation, small organic compounds, saccharides, carbohydrates,polysaccharides, lectins, peptides and analogs thereof, polypeptides,proteins, antibodies, oligonucleotides, polynucleotides, nucleic acids,etc. In one embodiment, the candidate compounds screened are smallorganic molecules having a molecular weight in the range of about100-2500 daltons. Such candidate molecules will often comprise cyclicalstructures composed of carbon atoms or mixtures of carbon atoms and oneor more heteroatoms and/or aromatic, polyaromatic, heteroaromatic and/orpolyaromatic structures. The candidate agents may include a wide varietyof functional group substituents. In one embodiment, the substituent(s)are independently selected from the group of substituents known tointeract with proteins, such as, for example, amine, carbonyl, hydroxyland carboxyl groups.

The candidate compounds may be screened on a compound-by-compound basisor, alternatively, using one of the myriad library techniques commonlyemployed in the art. For example, synthetic combinatorial compoundlibraries, natural products libraries and/or peptide libraries may bescreened using the assays of the invention to identify compounds thatmodulate Fcγ receptor-mediated mast or basophil cell activation.Although candidate compounds may be screened on a compound-by-compoundbasis, it may be more convenient to screen large numbers of candidatecompounds simultaneously using one of the many library screeningmethodologies known in the art. One art-known approach uses recombinantbacteriophage to produce large libraries of peptides that can then bescreened in a variety of formats. Using such phage methods, very largelibraries of candidate peptides can be constructed (e.g., 10⁶-10⁸peptides). Methods for constructing and screening such “phage display”libraries are described, for example, in Scott and Smith, Science249:386-390 (1990); Cwirla, S. E. et al., Proc. Natl. Acad. Sci. USA87:6378-6382 (1990); Devlin, J. J. et al., Science 249:404-406 (1990);U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,432,018; U.S. Pat. No.5,580,717 and U.S. Pat. No. 5,723,286, the disclosures of which areincorporated herein by reference. Other non-limiting examples ofrecombinant library methodologies that may be used in connection withthe assays of the invention are described in U.S. Pat. No. 6,156,571;U.S. Pat. No. 6,107,059 and U.S. Pat. No. 5,733,731, the disclosures ofwhich are incorporated herein by reference.

A second art-known approach uses chemical methods to synthesizelibraries of compounds, such as small organic compounds, peptides and/orpeptide analogs, attached to beads or wafers that can then beconveniently screened. The libraries may be encoded or non-encoded.Methods of synthesizing such immobilized libraries, as well as methodsof screening the libraries are described, for example, in Houghten,Proc. Natl. Acad. Sci. USA 82:5131-5735 (1985); Geysen et al., MolecularImmunology 23:709-715 (1986); Geysen et al., J. Immunologic Method102:259-274 (1987); Frank and Döring, Tetrahedron 44:6031-6040 (1988);Fodor et al., Science 251:767-773 (1991); Furka et al., 4thInternational Congress of Biochemistry, Volume 5, Abstract FR:013(1988); Furka, Int. J. Peptide Protein Res. 37:487-493 (1991); Frank,Tetrahedron 48:9217-9232 (1991); Needels et al., Proc. Natl. Acad. Sci.USA 90:10700-10704 (1993); DeWitt et al., Proc. Natl. Acad. Sci. USA90:6909-6913 (1993); Frank et al., Biorg. Med. Chem. Lett. 3:425-430(1993); Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 90:10922-10926(1993); WO 92/00252; WO 9428028; U.S. Pat. No. 6,329,143; U.S. Pat. No.6,291,183; U.S. Pat. No. 5,885,837; U.S. Pat. No. 5,424,186; U.S. Pat.No. 5,384,261; U.S. Pat. No. 6,165,717; U.S. Pat. No. 6,143,497; U.S.Pat. No. 6,140,493; U.S. Pat. No. 5,789,162; U.S. Pat. No. 5,770,358;U.S. Pat. No. 5,708,153; U.S. Pat. No. 5,639,603; U.S. Pat. No.5,541,061; U.S. Pat. No. 5,525,735; U.S. Pat. No. 5,525,734; U.S. Pat.No. 6,261,776; U.S. Pat. No. 6,239,273; U.S. Pat. No. 5,846,839; U.S.Pat. No. 5,770,455; U.S. Pat. No. 5,770,157; U.S. Pat. No. 5,609,826;U.S. Pat. No. 6,001,579; U.S. Pat. No. 5,968,736; U.S. Pat. No.5,962,337; U.S. Pat. No. 5,789,172; U.S. Pat. No. 5,721,099; U.S. Pat.No. 5,565,324; U.S. Pat. No. 5,010,175; and U.S. Pat. No. 4,631,211, thedisclosures of which are incorporated herein by reference. For reviewsof some of these techniques, see Ellman et al., Account, Chem. Res.29:132-143 (1996); Gallop et al., J. Med. Chem. 37:1233-1251 (1994);Gordon et al., J. Med. Chem. 37:1385-1401 (1994). Non-limiting examplesof solid-phase chemical synthesis strategies and conditions useful forsynthesizing combinatorial libraries of small organic and othercompounds may be found in Bunin, The Combinatorial Index, AcademicPress, London, England (1998) (see, e.g., Chapters 1-5) and Hemikens etal., Tetrahedron 52:4527-4554 (1996), as well as the references citedtherein, the disclosures of which are incorporated herein by reference.

Another art-known approach utilizes solution-phase chemical synthesistechniques to synthesize libraries of compounds, such as, for example,libraries of small organic compounds, which may then be screened in theassays of the invention. Methods for synthesizing and screening suchsolution-phase libraries are well-known and are described, for example,in Bunin, The Combinatorial Index, Academic Press, England (1998) (see,e.g., Chapter 6); WO 95/02566; U.S. Pat. No. 5,962,736; U.S. Pat. No.5,766,481; U.S. Pat. No. 5,736,412 and U.S. Pat. No. 5,712,171, and thereferences cited therein; the disclosures of which are incorporatedherein by reference. Additional review articles, references, patents andbooks describing myriad techniques for synthesizing and screeninglibraries of compounds can be found at Lebl and Leblova: DynamicDatabase of References in Molecular Diversity, Internet www.5z.com (seeespecially the diversity information pages at www.5z.com/divinfo).

The mast or basophil cell may be contacted with the candidate compoundsbefore, during, or after activating the Fcγ receptor signaling pathway.Preferably, in methods of identifying compounds that modulate Fcγreceptor signaling cascade, the cells are contacted with the candidatecompounds prior to activation of the signaling pathway and/or inpresence of Fcγ receptor activation. In some instances, it is desirableto determine the effect of the candidate compounds on an unactivated,primed or unprimed cell, or where activation is inhibited. In methods ofidentifying compounds that modulate IgE-mediated priming of cells, themast or basophil cells are preferably contacted with candidate compoundsprior to priming the cells with IgE, to distinguish effects of thecompound on priming versus cell activation.

The candidate agent can be added exogenously to the cells or can beadministered into the cells. For compounds that readily traverse cellmembranes, the compound may be administered to the cell by contactingthe cell with the compound. In one embodiment, such compounds may beadministered using well-known retroviral vectors and infectiontechniques pioneered by Richard Mulligan and David Baltimore with Psi-2lines and analogous retroviral packaging systems based upon NIH 3T3cells (see Mann et al., Cell 33:153-159 (1993), the disclosure of whichis incorporated herein by reference). Such helper-defective packagingcell lines are capable of producing all of the necessary trans proteins(gag, pol and env) required for packaging, processing, reversetranscribing and integrating genomes. Those RNA molecules that have incis the ψ packaging signal are packaged into maturing retrovirions.Virtually any of the art-known retroviral vectors and/or transfectionsystems may be used. Specific non-limiting examples of suitabletransfection systems include those described in WO 97/27213; WO97/27212; Choate et al., Human Gene Therapy 7:2247-2253 (1996); Kinsellaet al., Human Gene Therapy 7:1405-1413 (1996); Hofmann et al., Proc.Natl. Acad. Sci. USA 93:5185-5190 (1996); Kitamura et al., Proc. Natl.Acad. Sci. USA 92:9146-9150 (1995); WO 94/19478; Pear et al., Proc.Natl. Acad. Sci. USA 90:8392-8396 (1993); Mann et al., Cell 33:153-159(1993) and the references cited in all of the above, the disclosures ofwhich are incorporated herein by reference. Specific non-limitingexamples of suitable retroviral vector systems include vectors basedupon murine stem cell virus (MSCV) as described in Hawley et al., GeneTherapy 1:136-138 (1994); vectors based upon a modified MFG virus asdescribed in Rivere et al., Genetics 92:6733 (1995); pBABE as describedin WO 97/27213 and WO 97/27212; and the vectors depicted in FIG. 11 ofWO 01/34806, the disclosures of which are incorporated herein byreference. Other suitable vectors and transfection techniques, orcarrier systems for administration into cells, including methods such ascalcium phosphate, DEAE dextran, liposomes, electroporation, biolisticparticle bombardment, micro- and nanoparticles, and the like, are wellknown in the art.

Ideally, the ability to inhibit the release of all desired types ofmediators will reside in a single compound. However, mixtures ofcompounds can also be identified that achieve the same result. Forexample, a first compound which inhibits release of granule specificmediators may be used in combination with a second compound whichinhibits the release and/or synthesis of cytokine mediators.

In another embodiment, the method is used to identify candidatecompounds that may modulate, in particular to inhibit, Fcγreceptor-mediated mast or basophil cell activation as a therapeuticapproach towards the treatment or prevention of diseases characterizedby, caused by and/or associated with the release or synthesis ofchemical mediators of Fcγ receptor signaling cascades or degranulation.Diseases that are characterized by, caused by or associated with suchmediator release, synthesis or degranulation, and that can therefore betreated or prevented with the candidate compounds include, by way ofexample and not limitation, atopy or anaphylactic hypersensitivity orallergic reactions, allergies (e.g., allergic conjunctivitis, allergicrhinitis, atopic asthma, atopic dermatitis and food allergies), lowgrade scarring (e.g., of scleroderma, increased fibrosis, keloids,post-surgical scars, pulmonary fibrosis, vascular spasms, migraine,reperfusion injury and post myocardial infarction), diseases associatedwith tissue destruction (e.g., of COPD, cardiobronchitis and postmyocardial infarction), diseases associated with tissue inflammation(e.g., irritable bowel syndrome, spastic colon and inflammatory boweldisease), inflammation and scarring.

Kits

The invention also provides kits for carying out the various screeningassays and methods of the invention. These kits will typically includeIgE antibodies for priming cells; the components required for activatingthe mast or basophil cells, for example F(ab)₂ directed against FcγRIreceptor (particularly human FcγRI) and anti-F(ab)₂ antibodies forcrosslinking and receptor activation; and populations of mast and/orbasophil cells. The kit may further include components for detectingcell activation, including, substrates for granule-associated proteases(e.g., tryptase or chymase) or enzymes (e.g., hexosaminidase), or ELISAassays for late stage mediators (e.g., leukotrienes and cytokines).Additional components of the kits, include as non-limiting examples,labels, cell culture media, buffers, etc.

The invention having been described, the following examples are offeredby way of illustration and not limitation. They are not intended to beexhaustive or to limit the invention to the precise forms disclosed, andobviously many modifications and variations are possible in light of theabove teaching.

All patents, patent applications, publications, and references citedherein are expressly incorporated by reference to the same extent as ifeach individual publication or patent application was specifically andindividually indicated to be incorporated by reference.

EXAMPLES Example 1 Culturing of Human Mast and Basophil Cells

Human mast and basophil cells were cultured from progenitor cells asdescribed below (see also the methods described in copending U.S.application Ser. No. 10/053,355, filed Nov. 8, 2001, the disclosure ofwhich is incorporated herein by reference).

Preparation of STEMPRO-34 Complete Medium

To prepare STEMPRO-34 complete medium (“CM”), 250 mL STEMPRO-34TM serumfree medium (“SFM”; GibcoBRL, Catalog No. 10640) was added to a filterflask. To this was added 13 mL STEMPRO-34 Nutrient Supplement (“NS”;GibcoBRL, Catalog No. 10641) (prepared as described in more detail,below). The NS container was rinsed with approximately 10 mL SFM and therinse added to the filter flask. Following addition of 5 mL L-glutamine(200 mM; Mediatech, Catalog No. MT 25-005-CI and 5 mL 100×penicillin/streptomycin (“pen-strep”; HyClone, Catalog No. SV30010), thevolume was brought to 500 mL with SFM and the solution was filtered.

The most variable aspect of preparing the CM is the method by which theNS is thawed and mixed prior to addition to the SFM. The NS should bethawed in a 37° C. water bath and swirled, not vortexed or shaken, untilit is completely in solution. While swirling, it should be noted whetherthere are any lipids that are not yet in solution. If lipids are presentand the NS is not uniform in appearance, NS is returned to the waterbath and the swirling process repeated until it is uniform inappearance. Sometimes this component goes into solution immediately,sometimes after a couple of swirling cycles, and sometimes not at all.If, after a couple of hours, the NS is still not in solution, it isdiscarded and a fresh unit is used. NS that appears non-uniform afterthaw should not be used.

Expansion of CD34+ Cells

A starting population of CD34-positive (CD34+) cells of relatively smallnumber (1-5×10⁶ cells) was expanded to a relatively large number ofprogenitor cells (about 2-4×10⁹ cells) using the culture media andmethods described below. The CD34+ cells (from a single donor) wereobtained from Allcells (Berkeley, Calif.). Because there is a degree ofvariation in the quality and number of CD34+ cells that Allcellstypically provides, the newly delivered cells were transferred to a 15mL conical tube and brought up to 10 mL in CM prior to use.

On day 0, a cell count was performed on the viable (phase-bright) cellsand the cells were spun at 1200 rpm to pellet. The cells wereresuspended to a density of 275,000 cells/mL with CM containing 200ng/nL recombinant human Stem Cell Factor (“SCF”; Peprotech, Catalog No.300-07) and 20 ng/mL human flt-3 ligand (Peprotech, Catalog No. 300-19)(“CM/SCF/flt-3 medium”). On about day 4 or 5, the density of the culturewas checked by performing a cell count and the culture was diluted to adensity of 275,000 cells/mL with fresh CM/SCF/flt-3 medium. On about day7, the culture was transferred to a sterile tube and a cell count wasperformed. The cells were spun at 1200 rpm and resuspended to a densityof 275,000 cells/nL with fresh CM/SCF/flt-3 medium.

This cycle was repeated, starting from day 0, a total of 3-5 times overthe expansion period.

When the culture is large and being maintained in multiple flasks and isto be resuspended, the contents of all of the flasks are combined into asingle container prior to performing a cell count. This ensures that anaccurate cell count is achieved and provides for a degree of uniformityof treatment for the entire population. Each flask is checked separatelyfor contamination under the microscope prior to combining to preventcontamination of the entire population.

Between days 17-24, the culture can begin to go into decline (i.e.,approximately 5-10% of the total number of cells die) and fail to expandas rapidly as before. The cells are then monitored on a daily basisduring this time, as complete failure of the culture can take place inas short as 24 hours. Once the decline has begun, the cells are counted,spun down at 850 rpm for 15 minutes, and resuspended at a density of350,000 cells/mL in CM/SCF/flt-3 medium to induce one or two moredivisions out of the culture. The cells are monitored daily to avoidfailure of the culture.

When greater than 15% cell death is evident in the progenitor cellculture and some debris is present in the culture, the progenitor cellsare ready to be differentiated.

Differentiation of Progenitor Cells into Mucosal Mast Cells

A second phase is performed to convert the expanded progenitor cellsinto differentiated mucosal mast cells. These mucosal cultured humanmast cells (“CHMC”) are derived from CD34+ cells isolated from umbilicalcord blood and treated to form a proliferated population of progenitorcells, as described above. To produce the progenitor cells, theresuspension cycle for the culture was the same as that described above,except that the culture was seeded at a density of 425,000 cells/mL and15% additional media was added on about day four or five withoutperforming a cell count. Also, the cytokine composition of the mediumwas modified such that it contained SCF (200 ng/mL) and recombinanthuman IL-6 (200 ng/mL; Peprotech, Catalog No. 200-06 reconstituted to100 ug/mL in sterile 10 mM acetic acid) (“CM/SCF/IL-6 medium”).

Phases I and II together span approximately 5 weeks. Some death anddebris in the culture is evident during weeks 1-3 and there is a periodduring weeks 2-5 during which a small percentage of the culture is nolonger in suspension, but is instead attached to the surface of theculture vessel.

As during Phase I, when the culture is to be resuspended on day seven ofeach cycle, the contents of all flasks are combined into a singlecontainer prior to performing a cell count to ensure uniformity of theentire population. Each flask is checked separately for contaminationunder the microscope prior to combining to prevent contamination of theentire population.

When the flasks are combined, approximately 75% of the volume istransferred to the communal container, leaving behind about 10 mL or soin the flask. The flask containing the remaining volume was rappedsharply and laterally to dislodge the attached cells. The rapping wasrepeated at a right angle to the first rap to completely dislodge thecells.

The flask was leaned at a 45 degree angle for a couple of minutes beforethe remaining volume was transferred to the counting vessel. The cellswere spun at 950 rpm for 15 min prior to seeding at 35-50 mL per flask(at a density of 425,000 cells/mL).

Differentiation of Progenitor Cells into Connective Tissue-Type MastCells

A proliferated population of progenitor cells is prepared as above andtreated to form a tryptase/chymase positive (connective tissue)phenotype. The methods are performed as described above for mucosal mastcells, but with the substitution of IL-4 for IL-6 in the culture medium.The cells obtained are typical of connective tissue mast cells.

Differentiation of Progenitor Cells into Basophil Cells

A proliferated population of progenitor cells is prepared as describedabove, and used to form a proliferated population of basophil cells. Theprogenitor cells are treated as described for mucosal mast cells, butwith the substitution of IL-3 (at 20-50 ng/mL) for IL-6 in the culturemedium.

Example 2 Priming and Stimulation Conditions for FcγRI Activation ofCultured Human Mast Cells (CHMC)

The culture media contained the following components:

-   -   a) Gibco's StemPro-34 SFM Complete medium;        -   StemPro-34 SFM, Catalog No. 10640, 500 mL        -   StemPro-34 Nutrient Supplement, Catalog No. 10641, 13 mL    -   b) L-Glutamine: 200 mM Solution, Mediatech, Catalog No. MT        25-005-CI        -   add 5 mL per 500 mL StemPro    -   c) Penicillin/Streptomycin Soln. 100×, HyClone, Catalog No.        SV30010        -   add 5 mL per 500 mL StemPro.

Priming of mast cells is carried out by seeding the cells on Day 0 at3.5×10⁵ cells/mL in culture media plus SCF (200 ng/mL), IL-6 (200ng/mL), IL-4 (20 ng/nL), IgE (200 ng/mL), and IgG3 (200 ng/mL).

Activation or stimulation of cells is done on Day 3 by centrifuging thecells down and resupending them at 1.5×10³ cells/mL in modified tyrodes(MT: 137 mM NaCl, 2.7 mM KCI, 1.8 mM CaCl₂, 5.6 mM glucose, 20 mM Hepes(pH 7.4), 0.1% Bovine Serum Albumin (Sigma A4503)), with or withoutrabbit anti-human IgG (200 ng/mL final conc.), for 30 minutes at 37° C.(see Demo, S. D., Cytometry. 36(4):340-348 (1999)).

Degranulation is measured by centrifuging down the sample, andharvesting the supernatant and measuring tryptase activity. The tryptaseassays are performed under standard conditions (25 uL supernatant addedto 100 uL of 10 uM tryptase substrate [20 mM stock solution ofZ-Ala-Lys-Arg-AMC2TFA (Enzyme systems Products, Catalog No. AMC-246)diluted 1:2000 in tryptase assay buffer [0.1 MM Hepes (pH 7.5), 10% w/vGlycerol, 10 eM Heparin, 0.01% NaN₃] for 30 min at 37° C. The reactionsare read in plate reader (Wallac, Victor 2, 1420 Multilabel Counter).

Example 3 Screening of Small Molecule Candidate Compounds for Modulationof CHMC IgE and/or IgG Activation

To duplicate 96-well U-bottom plates (Costar 3799) are added 65 ul ofcompound dilutions or control samples prepared in MT [137 mM NaCl,2.7 mMKCL 1.8 MM CaCl₂, 5.6 mM glucose, 20 mM Hepes (pH 7.4), 0.1% BovineSerum Albumin (Sigma A4503)] containing 2% MeOH and 1% DMSO, and primedwith IgE as in Example 2. The mast cell modulating compounds comprise2,4-pyrimidinediamine compounds disclosed in U.S. patent applicationSer. No. 10/355,543, hereby incorporated by reference. CMHC cells arepelleted (980 rpm, 10 min) and resuspended in warm MT. To each 96-wellplate is added 65 ul of cells (1000-1500 cells/well). After mixing fourtimes, the cells are incubated for 1 hr at 37° C. and then stimulatedwith anti-IgE and/or anti-IgG. For controls, MT is added to wellscontaining unstimulated cells. Stimulation is for 30 minutes at 37° C.Assay for tryptase activity is carried out as above. Leukotriene C4 inthe supernatant is quantified using ELISA kit (Cayman Chemical Co., AnnArbor, Mich.; Catalog No. 520211) following supplier's instructions.

1. A method of identifying a compound capable of modulating Fcγ receptorsignaling pathway, comprising a) contacting at least one IgE primed mastcell with a candidate compound in the presence of Fcγ receptor signalingactivation; and b) determining whether the candidate compound modulatesthe Fcγ receptor-mediated signaling cascade.
 2. The method of claim 1 inwhich the candidate compound inhibits the Fcγ receptor signalingcascade.
 3. The method of claim 1 in which the Fcγ receptor is FcγRI. 4.The method of claim 1 in which the mast cell is a cultured mast cell. 5.The method of claim 1 in which the mast cell is a mucosal mast cell. 6.The method of claim 1 in which the mast cell is a human mast cell. 7.The method of claim 1 in which the modulation of Fcγ receptor signalingcascade is determined by measuring degranulation.
 8. The method of claim7 in which the modulation of Fcγ receptor signaling is determined bycomparing Fcγ receptor-mediated mast cell activation in presence andabsence of the candidate compound.
 9. The method of claim 1 in which thecompound is a small organic compound.
 10. The method of claim 9 in whichthe small organic compound has a molecular weight in the range of about100-2500 daltons.
 11. A method of identifying a compound for treatingdisorders of IgG-mediated mast cell activation, comprising: a)contacting at least one IgE primed mast cell with a candidate compoundin the presence of Fcγ receptor signaling activation; and b) determiningwhether the candidate compound modulates the Fcγ receptor signalingcascade.
 12. The method of claim 11 in which the candidate compoundinhibits the Fcγ receptor signaling cascade.
 13. The method of claim 11in which the Fcγ receptor is FcγRI.
 14. The method of claim 11 in whichthe mast cell is a cultured mast cell.
 15. The method of claim 11 inwhich the mast cell is a mucosal mast cell.
 16. The method of claim 11in which the mast cell is a human mast cell.
 17. The method of claim 11in which modulation of the Fcγ receptor signaling cascade is determinedby measuring degranulation.
 18. The method of claim 11 in whichmodulation of the Fcγ receptor signaling cascade is determined bycomparing Fcγ receptor-mediated mast cell activation in presence andabsence of the candidate compound.
 19. The method of claim 11 in whichthe compound is a small organic compound.
 20. The method of claim 19 inwhich the small organic compound has a molecular weight in the range ofabout 100-2500 daltons.
 21. A method of identifying a compound capableof modulating IgE priming of mast cells, comprising: a) contacting atleast one mast cell with a candidate compound and priming the mast cellwith IgE antibody, b) activating signal transduction via Fcγreceptor-mediated signaling pathway, and c) determining whether thecandidate compound modulates IgE priming of the mast cell.
 22. Themethod of claim 21 in which the candidate compound inhibits IgE primingof the mast cell.
 23. The method of claim 21 in which the Fcγ receptoris FcγRI.
 24. The method of claim 21 in which the mast cell is acultured mast cell.
 25. The method of claim 21 in which the mast cell isa mucosal mast cell.
 26. The method of claim 21 in which the mast cellis a human mast cell.
 27. The method of claim 21 in which modulation ofthe IgE priming of the mast cell is determined by measuringdegranulation.
 28. The method of claim 21 in which modulation of the IgEpriming of mast cell is determined by comparing IgE priming in presenceand absence of the candidate compound.
 29. The method of claim 21 inwhich the compound is a small organic compound.
 30. The method of claim29 in which the small organic compound has a molecular weight in therange of about 100-2500 daltons.